An anti-neutrophil cytoplasmic antibodies (ANCA), directed toward myeloperoxidase (MPO) and proteinase 3 (PR3), are detected in the majority of patients with pauci-immune necrotizing glomerulonephritis and small vessel vasculitis. The factors involved in the generation of these autoantibodies remain unknown. We hypothesize that the low incidence of ANCA and their associated diseases is in part due to requirements for particular V region gene recombinations, specific somatic mutations, or both and that the expression of anti-MP antibodies in mice is the result of a breakdown in tolerance. The study of anti-MPO antibodies derived from unimmunized SCG/KJ mice revealed a restriction in Vk gene use, and provides evidence that MPO is a driving antigen in the anti-MPO response. The two specific aims of this project are to (1) assess the affinity of murine anti-MPO autoantibodies, and (2) study the regulation of the anti-MPO response in mice. Aim 1 will be pursued by co-expressing anti-MPO derived H or L chains with a variety of non-anti-MPO-derived chains, and by introducing of removing specific somatic mutations using site-directed mutagenesis. The specificity and avidity of the resulting antibodies will be measured by ELISA and Biosensor based techniques. Aim 2 will be approached by the generation of mice transgenic for anti- MPO-light chain only, heavy chain only, and both. This approach will allow to study at which level of their maturation anti-MPO B cells are regulated. These two aims will help fill an important gap in our current knowledge of these mechanisms involved in the generation and regulation of the anti- MPO autoimmune responses.